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Versidad Autónoma De Puebla BENEMÉRITA UNIVERSIDAD AUTÓNOMA DE PUEBLA FACULTAD DE CIENCIAS BIOLÓGICAS Obtención del código de barras de ADN del gen MT-COI de Macrodactylus mexicanus y Macrodactylus nigripes (Coleoptera: Melolonthidae) Tesis que para obtener el título de LICENCIADO (A) EN BIOLOGÍA PRESENTA: ALBA GABRIELA GONZÁLEZ MARTÍNEZ DIRECTORA: MARÍA ROSETE ENRÍQUEZ CO-DIRECTOR: ANGEL ALONSO ROMERO LÓPEZ ABRIL 2018 Agradecimientos A mis padres Armando González Barbosa y Alba Martínez Rodríguez, porque ellos son la motivación de mi vida, por mostrarme el camino de la superación y siempre apoyarme. A mi hermano Francisco Armando González Martínez por su apoyo durante la carrera y por enseñarme a que trabajando y esforzándote puedes cumplir tus metas y crecer tanto en el ámbito personal y laboral. A mi directora de tesis María Rosete Enríquez por haberme brindado la oportunidad de realizar este trabajo, así como también por su tiempo dedicado. A mi co- director de tesis Angel Alonso Romero López por su ayuda, dedicación y asesoramiento. A los revisores por su valiosa colaboración para mejorar el escrito. ii Índice ABREVIATURAS GENERALES ................................................................................................... v LISTA DE FIGURAS ....................................................................................................................... vii LISTA DE CUADROS .................................................................................................................... viii RESUMEN ......................................................................................................................................... ix 1. Introducción .............................................................................................................................. 10 2. Antecedentes ......................................................................................................................... 11 2.1 Aspectos generales de sistemática y taxonomía ..................................................... 11 2.1.1 Taxonomía morfológica ............................................................................................... 13 2.1.2 Taxonomía molecular .................................................................................................. 13 2.1.3 Taxonomía integrativa ................................................................................................. 14 2.2 Código de barras de ADN ................................................................................................ 15 2.2.1 Propiedades del código de barras ............................................................................. 19 2.2.2 Código de barras en insectos ..................................................................................... 21 2.3 Generalidades de la familia Melolonthidae ................................................................. 23 2.3.1 Clasificación .................................................................................................................. 23 2.3.2. Caracteres morfológicos y distribución .................................................................... 25 2.4 Generalidades del género Macrodactylus .................................................................. 27 2.4.1 Descripción morfológica a nivel genérico ................................................................. 27 2.4.2 Descripción morfológica y hábitos de adultos y larvas de ..................................... 28 Macrodactylus mexicanus y Macrodactylus nigripes ........................................................ 28 3. Justificación .............................................................................................................................. 33 4. Hipótesis .................................................................................................................................... 34 5. Objetivos .................................................................................................................................... 34 5.1 Objetivo general ................................................................................................................. 34 5.2 Objetivos particulares .......................................................................................................... 34 5.2.1 Identificar adultos de M. mexicanus y M. nigripes mediante la revisión de caracteres morfológicos externos e internos. .................................................................... 34 5.2.2 Obtener la secuencia parcial del gen MT-COI a partir de muestras de ADN genómico de adultos de M. mexicanus y M. nigripes. ...................................................... 34 5.2.3. Determinar el porcentaje de similitud entre secuencias parciales del gen MT- COI por medio de herramientas bioinformáticas. .............................................................. 34 6. Material y métodos .................................................................................................................. 35 iii 6.1 Recolecta de adultos ........................................................................................................ 35 6.2 Identificación con caracteres morfológicos a través de claves dicotómicas ... 35 6.3 Identificación por taxonomía molecular con la obtención del código de barras de ADN del gen MT-COI .......................................................................................................... 36 6.3.1 Purificación del DNA .................................................................................................... 36 6.3.2 Amplificación del gen MT-COI por PCR punto final con iniciadores universales 37 6.3.3 Secuenciación y análisis bioinformáticos ................................................................. 38 6.3.4 Análisis de secuencias ................................................................................................ 38 7. Resultados ................................................................................................................................. 39 7.1 Identificación por taxonomía morfológica .................................................................. 39 7.1.1 Montaje de especímenes ............................................................................................ 43 7.2 Identificación por taxonomía molecular ...................................................................... 45 7.2.2 Obtención del código de barras del gen MT-COI ................................................... 46 7.2.3 Registro en base de datos de códigos de barras .............................................. 49 7.2.4 Análisis de secuencias homólogas del gen MT-COI ........................................ 52 8. Discusión ................................................................................................................................... 54 9. Conclusiones ............................................................................................................................ 58 10. Literatura citada ..................................................................................................................... 59 iv ABREVIATURAS GENERALES ADN: ácido desoxirribonucleico AGIMP: Proyecto General de Insectos de importancia agrícola en la India ARN: ácido ribonucleico ARNr: ácido ribonucleico ribosomal ARNt: ácido ribonucleico de transferencia BOLD: The Barcode of Life Data Systems BLAST: Basic Local Alignment Search Tool BLASTN: Nucleotide- Nucleotide BLAST CAT: catálogos CBOL: The Consortium for the Barcode of Life COI: citocromo oxidasa 1 dNTPs: mezcla de cuatro desoxirribonucleótidos trifosfato EF-1α: factor de elongación alpha 1 ITS2: región espaciadora interna transcrita 2 MT-COI: gen mitocondrial citocromo oxidasa 1 matK: gen de la mutarasa PCGs: genes codificadores de proteínas PCR: reacción en cadena de la polimerasa PMI: intervalo post mortem rbcL: gen del cloroplasto que codifica para RuBisCO tufA2: factor de elongación Tu h: horas min: minutos s: segundos ml: mililitros µl: microlitros rpm: revoluciones por minuto nm: nanómetros v: volts v pb: pares de bases Figura 2. ATP6: adenosin trifosfato subunidad 6 ATP8: adenosin trifosfato subunidad 8 cob: citocromo oxidasa b cox1-3: citocromo oxidasa c subunidad 1-3 CR: región de control nad1-6: NADH deshidrogenasa subunidad 1-6 nad4L: NADH deshidrogenasa subunidad 4L rrnS: subunidades de ARN ribosomal pequeñas rrnL: subunidades de ARN ribosomal grandes Figura 13. V1-3: Voucher 1-3 Cuadro 2. MmV1-3: Macrodactylus mexicanus voucher 1-3 Cuadro 3 MnV1-3: Macrodactylus nigripes voucher 1-3 vi LISTA DE FIGURAS Figura 1. Esquema general del mecanismo para obtener un código de barras de la vida Figura 2. Genoma mitocondrial de insecto Figura 3. Adultos de algunas de las primeras especies de Melolonthidae Figura 4. Ilustración de la maza antenal de Phyllophaga lissopyge Figura 5. Ilustración de la condición pleurosticti Figura 6. Ilustración morfológica de Macrodactylus mexicanus Figura 7. Ilustración morfológica de las estructuras genitales del macho y hembra de Macrodactylus mexicanus Figura 8. Ilustración morfológica de Macrodactylus nigripes Figura 9. Ilustración morfológica de las estructuras genitales del macho y hembra de Macrodactylus nigripes Figura 10. Imagen representativa de un macho de Macrodactylus mexicanus. Figura 11. Imagen representativa de un macho de Macrodactylus nigripes Figura 12. Montaje de Macrodactylus mexicanus y Macrodactylus nigripes Figura 13. Electroforesis en gel de agarosa de la amplificación
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